The influenza virus is an RNA envelop virus having a particle size of about 100 nm in diameter belonging to the family Orthomyxoviridae. It occurs in three types classified according to the antigenicity of internal protein thereof: types A, B and C. The influenza virus consists of an internal nucleocapsid surrounded by a viral envelop having a lipid bilayer structure or a ribonucleic acid (RNA) core associated with nuclear protein and an external glycoprotein. The inner layer of the viral envelop is configured mainly by matrix protein, whereas the outer layer is mostly configured by a host-derived lipid substance. The RNA of influenza virus assumes a segmentary structure. The influenza that spreads widely all over the world is caused by type A influenza viruses. Type A viruses have two kinds of envelop glycoproteins, i.e., hemagglutinin (HA) and neuraminidase (NA). According to antigenicity variation, HA is classified into 16 subtypes, and NA into 9 subtypes.
In recent years, highly pathogenic H5N1 avian influenza virus has been rampant worldwide; it could even be said that a new viral strain that can be communicated from one person to another could emerge and cause a pandemic at any moment. To cope with this situation, a global viral testing system is being enhanced, and large stockpiling of Tamiflu and the like as therapeutic drugs, vaccine development, production, and stockpiling are being implemented. However, the situation stands while many issues remain to be clarified, including when and how it will emerge, whether Tamiflu and the like will be therapeutically effective in the event thereof, whether the vaccine developed will be effective, when and to whom it will be inoculated, and, more importantly, when to institute a state of high alert, and when to call off it. This is because we are going to encounter a situation that has never been experienced by human being, where vaccines and therapeutic drugs for pathogens and viruses that have not yet emerged must be stockpiled. A problem with vaccine development, in particular, resides in the fact that every year many mutations occur in the hemagglutinin gene on the influenza virus genome to cause an antigenic drift (change in antigenicity), which is thought to be the cause of epidemic prevalence. Therefore, inoculating a vaccine that does not match the prevailing subtype does not have an expected prophylactic effect. The reason why no attempts have been made to develop antibody therapeutic drugs (prophylactic drugs) for influenza virus is that it is feared that a medicine that has been developed with considerable effort is no longer useful because the virus that should otherwise be neutralized at the time of development has changed its nature due to an antigenic drift.
The present inventors screened a phage display human antibody library generated from a large number of B lymphocytes collected from one individual for twelve influenza virus strains of subtype H3N2 separated between 1968 and 2004, and found that the majority of clones exhibiting neutralizing activity were anti-hemagglutinin antibodies, and that they were roughly dividable into three groups: those that specifically neutralize viral strains separated in 1968-1973, viral strains separated in 1977-1993, and viral strains separated in 1997-2003 (non-patent document 1). Although this finding upsets the conventional common notion that it is meaningless to develop antibody therapeutic drugs (prophylactic drug) for influenza virus, phage antibody libraries have not been seriously investigated to date since combinations of a heavy chain and a light chain do not always reflect the in vitro environment and for other reasons.
Against this background, three research groups independently succeeded in isolating human monoclonal antibodies that neutralize subtype H5 influenza viruses (patent documents 1 and 2, non-patent documents 2-4). These antibodies were shown to exhibit neutralizing activity not only on subtype H5 influenza viruses, but also on other subtypes (e.g., subtype H1 and the like). However, while the 16 subtypes of hemagglutinin (H1-H16) are classified according to epitope into two major groups (Groups 1 and 2), these antibodies exhibited neutralizing activity only against Group 1 (e.g., subtypes H1, H2, H5, H6, H8, H9 and the like), and did not exhibit neutralizing activity on subtypes of influenza virus belonging to Group 2 (e.g., subtypes H3 and H7 and the like). That is, no anti-influenza virus antibody that exhibits a broad range of neutralizing activity beyond the bather of the two groups based on the sequence of hemagglutinin has been isolated or reported.
X-ray structural analysis has revealed the binding modes of these antibodies and hemagglutinin, making it evident that the 38-position amino acid of hemagglutinin has changed to asparagine in subtypes H3 and H7 in Group 2 and undergoes N-type sugar chain modification (non-patent documents 4 and 5). Furthermore, it has been reported that introducing an N-type sugar chain modification site into the 38-position of H5 caused the binding ability of the neutralizing antibody to decrease by 70% (non-patent document 5). It is also known that when an influenza virus having its hemagglutinin mutated escapes a neutralizing antibody, such mutations accumulate mainly in five regions within the hemagglutinin gene (A, B, C, D and E regions), which reportedly comprises a neutralizing epitope (non-patent documents 6 and 7). These findings suggest difficulty in acquiring a neutralizing antibody that acts beyond this barrier between the two groups.